Biophysical evolution of the receptor binding domains of SARS-CoVs (preprint)

With hundreds of coronaviruses (CoVs) identified in bats that are capable of infecting humans, it is important to understand how CoVs that affected the human population have evolved. Seven known coronaviruses have infected humans, of which three CoVs caused severe disease with high mortality rates: SARS-CoV emerged in 2002, MERS-CoV in 2012, and SARS-CoV-2 in 2019. Both SARS-CoV and SARS-CoV-2 belong to the same family, follow the same receptor pathway, and use their receptor binding domain (RBD) of spike protein to bind to the ACE2 receptor on the human epithelial cell surface. The sequence of the two RBDs is divergent, especially in the receptor binding motif (RBM) that directly interacts with ACE2. We probed the biophysical differences between the two RBDs in terms of their structure, stability, aggregation, and function. Since RBD is being explored as an antigen in protein subunit vaccines against CoVs, determining these biophysical properties will also aid in developing stable protein subunit vaccines. Our results show that despite RBDs having a similar three-dimensional structure, they differ in their thermodynamic stability. RBD of SARS-CoV-2 is significantly less stable than that of SARS-CoV. Correspondingly, SARS-CoV-2 RBD shows a higher aggregation propensity. Regarding binding to ACE2, less stable SARS-CoV-2 RBD binds with a higher affinity than more stable SARS-CoV RBD. In addition, SARS-CoV-2 RBD is more homogenous in terms of its binding stoichiometry towards ACE2, compared to SARS-CoV RBD. These results indicate that SARS-CoV-2 RBD differs from SARS-CoV RBD in terms of its stability, aggregation, and function, possibly originating from the diverse RBMs. Higher aggregation propensity and decreased stability of SARS-CoV-2 RBD warrants further optimization of protein subunit vaccines that use RBD as an antigen either by inserting stabilizing mutations or formulation screening.

Biophysical fitness landscape of the SARS-CoV-2 Delta variant receptor binding domain (preprint)

Among the five known SARS-CoV-2 variants of concern, Delta is the most virulent leading to severe symptoms and increased number of deaths. Our study seeks to examine how the biophysical parameters of the Delta variant correlate to the clinical observations. Receptor binding domain (RBD) is the first point of contact with the human host cells and is the immunodominant form of the spike protein. Delta variant RBD contains two novel mutations L452R and T478K. We examined the effect of single mutations as well as the double mutation on RBD expression in human Expi293 cells, RBD stability using urea and thermal denaturation, and RBD binding to angiotensin converting enzyme 2 (ACE2) receptor and to neutralizing antibodies using isothermal titration calorimetry. Delta variant RBD showed significantly higher expression compared to the wild-type RBD, and the increased expression is due to L452R mutation. Despite their non-conservative nature, none of the mutations significantly affected RBD structure and stability. All mutants showed similar binding affinity to ACE2 and to Class 1 antibodies (CC12.1 and LY-CoV016) as that of the wild-type. Delta double mutant L452R/T478K showed no binding to Class 2 antibodies (P2B-2F6 and LY-CoV555) and a hundred-fold weaker binding to a Class 3 antibody (REGN10987), and the decreased antibody binding is determined by the L452R mutation. These results indicate that the immune escape from neutralizing antibodies, rather than receptor binding, is the main biophysical parameter determining the fitness landscape of the Delta variant RBD and is determined by the L452R mutation.

Interplay between protein stability, binding to ACE2 and escape from neutralizing antibodies determines the natural selection of SARS-CoV-2 receptor binding domain variants (preprint)

Emergence of new SARS-CoV-2 variants has raised concerns at the effectiveness of vaccines and antibody therapeutics developed against the unmutated wild-type virus. It is thus important to understand the emergence of mutants from an evolutionary viewpoint to be able to devise effective countermeasures against new variants. We examined the effect of 12 most commonly occurring mutations in the receptor binding domain (RBD) on its expression, stability, activity, and antibody escape potential- some of the factors that may influence the natural selection of mutants. Recombinant proteins were expressed in human cells. Stability was measured using thermal denaturation melts. Activity and antibody escape potential were measured using isothermal titration calorimetry in terms of binding of RBD variants to ACE2 and to a neutralizing human antibody CC12.1, respectively. Our results show that variants differ in their expression levels, suggesting that mutations can impact the availability of proteins for virus assembly. All variants have similar or higher stability than the wild-type, implying that increased RBD stability might be another important factor in virus evolution. In terms of ACE2 binding, when compared to the wild-type, only 3 out of 7 expressed variants show stronger affinity, 2 have similar affinity, whereas the other 2 have weaker affinity, indicating that increased affinity towards ACE2 is an important but not the sole factor in the natural selection of variants. In terms of CC12.1 binding, when compared to the wild-type, 4 out of 7 variants have weaker affinity, 2 have a similar affinity, and 1 variant has a stronger affinity. Taken together, these results indicate that multiple factors contribute towards the natural selection of variants, and all of these factors have to be considered in order to understand the natural selection of SARS-CoV-2 variants. In addition, since not all variants can escape a given neutralizing antibody, antibodies to treat new variants can be chosen based on the specific mutations in that particular variant.